title: Quantification of Alefacept, an immunosuppressive fusion protein in human plasma using a protein analogue internal standard, trypsin cleaved signature peptides and liquid chromatography tandem mass spectrometry.
abstract: Quantitative analysis of a therapeutic protein through use of surrogate proteotypic peptides was evaluated for the measurement of Amevive (Alefacept) in human plasma using liquid chromatography tandem mass spectrometry. Signature peptides were obtained through in silico and iterative tuning processes to represent Alefacept for quantification. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with pH controlled at 5.1 and heat denaturation at 45°C followed by enzymatic digestion, dilution, and filtration. On-line extraction of the signature peptides was carried out using a Phenomenex Gemini C18 security guard column (4.0 mm × 2.0 mm) as a loading column and a Gemini C18 (100 mm × 2.1 I.D., particle size 5 μm) as the analytical (eluting) column. Tandem mass spectrometric detection was performed on a hybrid triple quadrupole linear ion trap equipped with electrospray ionization to positively ionize signature peptides for Alefacept and myoglobin. The method was linear for Alefacept (protein) concentrations between 250 and 10,000 ng/mL. Precision and accuracy for inter- and intra-assay for the lower limit of quantification was less than 20% (16.2 and 10.3, respectively). The method was validated according to current FDA guidelines for bioanalytical method validation.
authors.affiliation: Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Halquist
authors.forename: H Thomas
authors.initials: HT
authors.lastname: Karnes
mesh_terms: D000077944:Alefacept; D000595:Amino Acid Sequence; D000818:Animals; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009211:Myoglobin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D000077944:Alefacept